Immunofluorescent staining with anti-smooth or anti-striated muscle myosin was carried out for 30 minutes at room temperature (18-20 degrees C) on cultures of smooth muscle cells and fibroblasts from guinea-pig vas deferens, taenia coli and ureter, rabbit aorta and chicken gizzard and of cardiac muscle cells and fibroblasts from rat ventricle. With anti-smooth muscle myosin, smooth muscle cells showed an intense fluorescent staining in fine fibrils with an "interrupted" appearance running parallel to the longitudinal axis of the cell throughout the cytoplasm, and also in coarser, "non-interrupted" fibrils (termed here "attachment fibrils") concentrated at the surface of the cell adjacent to the glass coverslip. Fibroblasts in the same cultures showed similar, but much weaker, reactions. When anti-striated myosin was added to the smooth muscle cultures, staining of neither cell type was observed. In contrast, cardiac muscle cells in cultures of rat ventricle did not react anti-smooth muscle myosin, but gave bright fluorescent A-band staining with anti-striated myosin. Fibroblasts in the ventricle cultures were unreactive with anti-striated muscle myosin but gave the characteristic weak reaction with anti-smooth muscle myosin. Thus immunofluorescent stainig with anti-smooth muscle myosin is useful for distinguishing between isolated smooth muscle cells and fibroblasts in tissue culture.
Smooth muscle cells dispersed by low concentration of trypsin (0.125%) and grown in tissue culture will not bind antibodies against smooth muscle myosin added to the culture medium. These cells will attach, flatten and contract normally. When the trypsin concentration is raised to 0.25%, many of the attached cells will not flatten. Such round cells show uptake of the myosin antibody at the periphery and in the cytoplasm, using the indirect immunofluorescent technique. At a trypsin concentration of 1%, viable cells are no longer observed and all cells show uptake of myosin antibody. It is concluded from these experiments that above a crucial trypsin concentration, the membrane becomes altered sufficiently to permit the penetration of antibodies into the cell interior.
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